The Journal of Neuroscience, January 16, 2008 • 28(3):587–597 • 587
Development/Plasticity/Repair
Organization and Function of the Blood–Brain Barrier in
Drosophila
Tobias Stork,1 Daniel Engelen,1 Alice Krudewig,1 Marion Silies,1 Roland J. Bainton,2 and Christian Klämbt1
1
Institut für Neurobiologie, Universität Münster, D-48149 Münster Germany, and 2Department of Anesthesia and Perioperative Care, University of
California, San Francisco, California 94158-2517
The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many
functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and
other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood– brain barrier in
Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the
surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of
extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments
in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes
in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles
(i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the
blood– brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal blood–
brain barrier function.
Key words: Drosophila; blood– brain barrier; glial cells; septate junction; development; perineurial glia; subperineurial glia
Introduction
Any complex nervous system comprises two different cell types.
Whereas the different types of neurons perceive and integrate
information, glial cells set the stage to provide the environment
required for normal functionality of neurons (Freeman and
Doherty, 2006). Indeed increasingly complicated neuronal connectivity is linked to the relative number of glial cells (Granderath
and Klämbt, 1999; Lemke, 2001). Compared with ⬎50% in
mammals, only 10% of all neural Drosophila cells are of glial
nature making it a particularly good model system to study glial
development and function (Edenfeld et al., 2005; Freeman and
Doherty, 2006).
Previous work suggested surprising parallels between the molecular mechanisms instructing Drosophila and mammalian glial
differentiation. In both systems, glial differentiation is dependent
on regulation of pre-mRNA splicing. Mutations in the splicing
regulator Quaking lead to a dysmyelination phenotype in mice
(Sidman et al., 1964; Ebersole et al., 1996). Similarly, mutations in
the Drosophila ortholog how disrupt glial differentiation by afReceived June 11, 2007; revised Nov. 13, 2007; accepted Dec. 4, 2007.
This work was supported by grants from the Deutsche Forschungsgemeinschaft to C.K. M.S. acknowledges a
fellowship from the Boehringer Ingelheim foundation. We are grateful for the many fly stocks that were sent by the
Bloomington stock center. We are thankful to our colleagues who provided additional flies stocks used in this study:
R. Fehon for coracle and nervana2 alleles, A. Giangrande for gcm mutants, M. Gonzales-Gaitan for flp-out strains, B.
Altenhein for the fasIIGal4 (Mz507 ) strain, H. Aberle for the chaGal4 strain, and U. Thomas for UAS:EGFPDlgS97 flies.
Correspondence should be addressed to Christian Klämbt, Institut für Neurobiologie, Universität Münster,
Badestrasse 9, D-48149 Münster, Germany. E-mail: [email protected].
DOI:10.1523/JNEUROSCI.4367-07.2008
Copyright © 2008 Society for Neuroscience 0270-6474/08/280587-11$15.00/0
fecting splicing (Edenfeld et al., 2006). One of the splicing targets
of the HOW complex is neurexinIV, which encodes a septate
junction component homologous to the mammalian Caspr protein. Moreover, septate junctions formed by myelinating glia at
the paranodal junctions as well as those formed by Drosophila
glial cells rely on sets of evolutionary conserved membrane proteins: Caspr/NeurexinIV, Contactin, and Neurofascin155/Neuroglian (Baumgartner et al., 1996; Bhat et al., 2001; Girault and
Peles, 2002; Poliak and Peles, 2003; Faivre-Sarrailh et al., 2004;
Banerjee et al., 2006). Thus, not only the regulation of glial differentiation, but also the final realization of glial differentiation
appears to be directed by evolutionary conserved mechanisms.
Despite the growing knowledge on early gliogenesis and subsequent differentiation, we do, however, know amazingly little
about the normal morphology and function of the different glial
cell types found in a fly nervous system (Ito et al., 1995; Van De
Bor and Giangrande, 2002; Freeman and Doherty, 2006). Here,
we present a detailed morphological characterization of the different glial cell layers using electron microscopy and confocal
analyses of single-labeled glial cells. Inward from a dense outer
extracellular matrix is a perineurial sheath of astrocyte-like
shaped cells, which are able to divide throughout larval development. Below the perineurium are the septate junction forming
subperineurial glial cells. The organization of the more inner glial
layers slightly differs in the PNS and CNS. In the PNS, inner
wrapping glial cells insulate individual axons of the nerves at the
end of larval development, whereas in the CNS cortex and neuropile glia are found below the subperineurial glial cell layer
Stork et al. • Formation of Glial Barriers in Drosophila
588 • J. Neurosci., January 16, 2008 • 28(3):587–597
where they ensheath neuronal cell bodies, axonal tracts and the
dendritic compartments (Pereanu et al., 2005).
To assay the functionality of the blood– brain barrier, we determined the kinetics of dye uptake for small and large dextran
molecules in wild-type and several mutant animals, which suggested that the barrier function is mostly mediated by the subperineurial cells, whereas the other glial cell layers contribute to
barrier selectivity against much larger particles.
Materials and Methods
Genetics. The following fly strains were used in this study: gcmN7– 4 (Vincent et al., 1996); moody⌬17 (Bainton et al., 2005); nrxIV4304 (Baumgartner et al., 1996); nrxIVEP604; nrg14 (Bloomington Stock Center, Bloomington, IN); nrv2k13315 (Genova and Fehon, 2003); coracle5 (Lamb et al.,
1998); Df(3R)ED5020 to remove contactin (Faivre-Sarrailh et al., 2004);
sinuNWU7 (Wu et al., 2004); Df(1)RR79 to remove the Claudin CG6398;
Df(2R)M60E to remove the Claudin CG3770; Df(2R)nap2 to remove the
Claudin CG1298; Df(3R)Excel6192 to remove the Claudin CG6982;
pckEA97 (megatrachea) (Behr et al., 2003); 454 NeurexinIV:GFP, 125 Lachesin:GFP (Edenfeld et al., 2006); 173 nervana2:GFP (U. Lammel, unpublished observations); UAS:GFP; UAS:laminGFP; UAS:flp,
nervana2Gal4 (Bloomington stock center); pvr7.1 Viking:GFP (Olofsson
and Page, 2005); 43Gal4 (Bloomington stock center); c527Gal4 (Hummel et al., 2002); FasIIGal4 (Mz507 ) (B. Altenhein, personal communication); chaGal4 (Salvaterra and Kitamoto, 2001); and SPG:Gal4 (Bainton et al., 2005). All deficiencies were obtained from the Bloomington
stock collection. To label individual glial cells, we followed a flip-out
strategy (Struhl and Basler, 1993). The following sources of Flp recombinase were used: repo:Gal4 UAS:flp and a direct repo:flp fusion (Silies et
al., 2007). The following flipout constructs were used: UAS⬎CD2
yellow⫹⬎mCD8GFP and tub ⬎64⬎Gal4 (Wong et al., 2002). In addition
we generated Mosaic analysis with a repressible cell marker (MARCM)
clones (Lee and Luo, 1999) using repo:flp; repoGal4 UAS:actinGFP
FRT80/FRT80 Gal80.
Immunohistochemistry and electron microscopic analyses. Antibodies
[22C10, Fasciclin II (Fas II), Repo] were obtained from the Developmental Studies Hybridoma bank (Iowa City, IA). Anti-␤Gal (Cappel, Cochranville, PA), anti-GFP (Invitrogen, Eugene, OR), and anti-HRP Cy5
(Dianova, Hamburg, Germany) antisera were used according to the
manufacturer’s instructions. Fixation and treatment of tissues for immunohistochemistry was performed according to standard procedures. For
electron microscopic (EM) analyses, embryos were fixed in
glutaraldehyde-saturated N-heptane and the vitelline membrane was removed by hand. Stage 17 embryos were injected with 4% PFA and immediately opened on both ends. They were then fixed overnight in 4%
PFA. Larval tissues were also fixed in 4% PFA at 4°C overnight. After a
fixation in 2% OsO4 for 1 h at room temperature and 2% uranylacetate
treatment for 30 min at room temperature in the dark, tissues were
embedded in epon as described previously (Stollewerk et al., 1996;
Stollewerk and Klämbt, 1997), but acrolein treatment was omitted. Ultrathin sections were imaged with a Zeiss (Oberkochen, Germany)
EM900 with an SIS Morada digital camera. Fluorescently labeled specimens were analyzed using a Zeiss 510 LSM; orthogonal sections were
taken using the Zeiss image browser.
Dextran uptake. The following dextrans were used: 2.5 mM 10 kDa
Texas red conjugated dextran (Invitrogen) in H2O; 0.4 mM 70 kDa FITC
conjugated anionic dextran (Invitrogen) in H2O; and 0.01 mM 500 kDa
FITC conjugated anionic dextran (Invitrogen) in H2O. Dextran solutions were injected into stage 17 embryos according to standard procedures (Schwabe et al., 2005). The genotype of the embryos was determined using green fluorescent protein (GFP)-labeled balancer
chromosomes. To calculate the uptake of the fluorescence label into the
nervous system we generated stacks of confocal images (2 ␮m thick;
through the entire nervous system) at 2, 10, 20, and 30 min after injection
with a Zeiss 5 Live LSM (set at four frames per second with 512 ⫻ 512
resolution). The laser settings were identical throughout the experiments
and were controlled using a Convallaria majalis slide provided by Zeiss.
The mean density of pixel intensity (ranging from 0 to 255) reflecting the
dextran uptake was determined in an identical area of five injected embryos per genotype using the Zeiss LSM software. To determine the
uptake kinetics, the same region was measured over time.
Results
Formation of the blood– brain barrier
All Drosophila glial cells, except the midline glia, express the repo
gene (Xiong et al., 1994; Lee and Jones, 2005). Several repopositive glial cell layers cover the Drosophila nervous system. Two
distinct cell types, the perineurial and the subperineurial glia
form the outer cell layers. They separate axons and neuronal cell
bodies, which are surrounded by cortex and wrapping glia, from
the high potassium concentration in the hemolymph to allow
normal electrical conductance. To characterize individual glial
cells we followed either a flip-out strategy and expressed the Flp
recombinase in all glial cells of flies carrying different flip-out
constructs or we generated MARCM clones [using either a repoflp strain (Silies et al., 2007) or a repoGal4 UASflp strain]. These
procedures result in the random, but specific labeling of few glial
cells in every animal. The MARCM technology requires cell division to generate labeled cell clones whereas the flip-out technology can also be applied in postmitotic cells (Struhl and Basler,
1993; Lee and Luo, 1999). In previous reports these outer glial cell
layers were often considered as single morphological unit termed
surface glia, peripheral glia or perineurium and no comprehensive assignment of the morphological and functional diversity has
been made (Bellen et al., 1998; Leiserson et al., 2000; Sepp et al.,
2000; Bainton et al., 2005; Schwabe et al., 2005; Freeman and
Doherty, 2006).
The neural lamella
A dense network of extra cellular matrix, called neural lamella,
surrounds both the central and the peripheral nervous system
and can be detected by a protein trap insertion into the collagen IV
locus (Fig. 1 A). The neural lamella can be detected from embryonic stage 16 onwards (supplemental Fig. 1 A, B, available at www.jneurosci.org as supplemental material). No distinction can be
made between the matrix covering the CNS and peripheral nervous system, indicating that the neural lamella is a continuous,
relatively unstructured layer. In mutants with reduced hemocyte
motility no lamella is formed (supplemental Fig. 1C, available at
www.jneurosci.org as supplemental material) (Olofsson and
Page, 2005).
The perineurial layer
The perineurial glial cells are located just below the neural lamella. We did not identify a gene trap exclusively expressed in
these cells. However, the Gal4 driver c527 (Hummel et al., 2002)
shows preferential activity in this cell type as can be seen in orthogonal sections of third instar peripheral nerves (Fig. 1 B). Cells
within the perineurial glial cell layer of the CNS exhibit a star-like
shape with many thin cell protrusions within the perineurial layer
itself, which never invade into the neural tissue (Fig. 2 A, B). Based
on several MARCM clones comprising only perineurial glial cells
(Fig. 2 B), we conclude that these cells can divide considerably
during larval stages. In the PNS, only few perineurial glial cells are
found in the first instar stage. In third larval instar nerves, perineurial cells divide and cover the nerve. Here they appear elongated with fine cell processes (Fig. 3C,f, arrowhead).
The subperineurial layer
The second glial cell layer is formed by the subperineurial cells.
These cells specifically express the moody gene (Bainton et al.,
Stork et al. • Formation of Glial Barriers in Drosophila
J. Neurosci., January 16, 2008 • 28(3):587–597 • 589
1999) positive wrapping glial cells are found (Fig. 1 F, supplemental Fig. 2, available at www.jneurosci.org as supplemental material). These cells extend long and thin processes that wrap individual axons (Fig. 3 A, B,a– d). In a given section of a peripheral
nerve two to three glial cell processes can be detected (see Figs. 3,
6) (see below). In the CNS, the inner glial layer is more complex
and comprises cortex glia that insulate neuronal cell bodies as
well as the initial segments of the axons and neuropil glia that
ensheathes the axon fascicles and presumably contacts individual
synapses in dendritic compartments (Younossi-Hartenstein et
al., 2003; Pereanu et al., 2005). Further details on these glial cell
classes will be presented elsewhere.
Sensory and motor axons are individually wrapped in
the periphery
The PNS harbors two distinct axonal classes: the sensory axons
that project into the CNS and the motor axons that project outwards from the CNS to the muscle fibers in the periphery. To
address whether wrapping glial cells keep sensory and motor
components separate, we used either a Gal4 driver that is active in
most sensory neurons (43Gal4) and counterstained with anti-Fas
II antibodies to label motor axons (Fig. 4 A, C) or we used a gene
trap insertion into the fas II gene mimicking fas II gene expression
in all motoneurons (#397) (U. Lammel, unpublished observation) and then counterstained with anti-Futsch antibodies that
label all sensory axons and only few motor axons (Hummel et al.,
2000) (Fig. 4 B, D). Confocal analyses of such embryos indicate
that indeed the motor and sensory axons are kept in distinct
fascicles (Fig. 4 A–D). To test the organization of sensory and
motor axons in third instar larvae we used a fasGal4 driver to label
motor axons and a chaGal4 driver to visualize sensory axons (Fig.
4 E, F ). Again, confocal analyses demonstrated a clear separation
of the two modalities within the segmental nerves.
Figure 1. Anatomy of the peripheral nerve. Orthogonal sections through stacks of confocal
images of nerves of third instar larvae stained for HRP expression (blue), Repo expression (red),
and GFP expression (green). A, The GFP-gene trap insertion in Collagen IV (Viking) labels the
neural lamella. B, The Gal4 driver strain c527 activates expression of CD8:GFP predominantly in
the perineurial cells, which are just below the neural lamella. C, The SPG-Gal4 driver activates
CD8:GFP expression in the subperineurial cells that tightly encircle the axonal fascicles (blue).
Note the occurrence of a glial cell nucleus in the fascicle (B, C). D, The GFP-gene trap insertion in
neurexinIV (#454 ) labels a thin stripe along the entire axonal fascicle. E, Repo-Gal4 activates
CD8:GFP expression in all glial cells. Note, that some GFP expression is also visible within the
nerve. F, A GFP-gene trap insertion in the nervana2 gene labels glial cell membranes within the
fascicle. Scale bars: 2 ␮m.
2005; Schwabe et al., 2005) and we used moody promoter sequences to generate the subperineurial glia (SPG)-Gal4 driver
(Fig. 1C). Generally, the subperineurial cells form a thin layer
below the perineurial cells (Figs. 1C, 2 D, F ). In the CNS, single
subperineurial cells labeled by the flip-out technique appear always as very large, hexagonally arranged cells often extending
40 – 80 ␮m in diameter (Fig. 2C–E). Subperineurial cells in the
PNS are very large as well and can form autocellular junctions
(see below).
The wrapping glia
The final glial cell layer in the PNS comprises the wrapping glia.
During late embryonic and early larval stages, these glial cells
initially contact fascicles of sensory and motor axons and only
later during the third larval stage ensheath almost every single
axon resembling the appearance of Schwann cells in Remak bundles of the mammalian PNS (see below) (Nave and Salzer, 2006).
Along the peripheral nerves, only three nervana2-Gal4 (Sun et al.,
Ultrastructural analysis
To corroborate the above findings we determined the ultrastructural appearance of the different glial cells at embryonic
and larval stages. At the electron microscopic level, the structure of the neural lamella appeared identical in the CNS and
the PNS (Fig. 5). In embryonic stages, only few perineurial
cells were identified. They generally develop numerous processes that strictly stay within the glial layer and mediate extensive cell– cell contacts among the perineurial cells (Fig.
5C,D). In contrast to later developmental stages, the perineurial cells do not cover the entire circumference of the nervous
system (Figs. 5 A, F,G, 6).
Below the perineurial cells lies the subperineurium. Electron
microscopic analyses confirm the flat cell shape of the subperineurial glia and demonstrate that these cells establish septate
junctions with each other (Figs. 5E, 6 A, inset) (see below). In the
CNS, the subperineurial cells are not in contact with axons, which
instead are wrapped by neuropile glia. In the PNS of the first
instar larvae, however, the subperineurial glial cells can be in
direct contact with axons as they form a sheath around the entire
nerve (Fig. 5 F, G).
In late embryos, the wrapping glia separates sensory and
motor axon fascicles. The wrapping glia has not yet started to
ensheath individual axons (Fig. 5 A, B). In first instar larvae,
the wrapping glia starts to ensheath individual peripheral axons (Fig. 5 F, G). During the third instar larvae stage, the wrapping glia has individually ensheathed most axons and the contact between axons and subperineurial cells is nearly
completely lost (Fig. 6).
Stork et al. • Formation of Glial Barriers in Drosophila
590 • J. Neurosci., January 16, 2008 • 28(3):587–597
In the periphery, the subperineurial glial
cells form autocellular junctions
Septate junctions formed by the subperineurial cells have long been considered to
be the structural basis for the blood– brain
barrier (Treherne, 1962; Treherne and Pichon, 1972; Carlson et al., 2000). Injection
of electron opaque tracer molecules demonstrated that these molecules could easily
penetrate through the neural lamella, but
did not progress beyond the junctional
complexes (Lane and Treherne, 1972).
More recently, it was shown that the
G-protein coupled receptor Moody is expressed specifically by subperineurial glial
cells and that loss of moody function results
in both a reduced formation of septate
junctions and a leaky blood– brain barrier
(Bainton et al., 2005; Schwabe et al., 2005).
In support of this we found septate
junctions prominently in the subperineurial glial layer in serial EM sections in both,
the CNS and the PNS (Figs. 5, 6; supplemental Fig. 3, available at www.jneurosci.org as supplemental material). Tracking
of glial cell membranes in serial electron
microscopic cross sections of peripheral
nerves shows that generally one subperineurial cell is detected which exhibits
prominent autocellular septate junctions
(Fig. 6). To further support the notion that
the subperineurial glial cells generate septate junctions we expressed a GFP-tagged
Discs large protein that in epithelial cells
faithfully localizes to septate junctions. After expression of the GFP::DlgS97 fusion
(Bachmann et al., 2004) in the subperineurial glial cell layer (using the SPG-Gal4
driver), the GFP::DlgS97 fusion protein
was recruited into septate junction-like
structures that resemble those labeled by
the neurexinIV gene trap insertion (Figs.
6 B, C, 7). In the periphery, septate junctions are visualized as a thin single line
closely following the axonal fascicles, and
only rarely we noted ring-like structures
around the nerve representing contact
zones of two subperineurial glial cells (Fig.
7 A, B), resembling the junctional organization in trachea (Fig. 7C) (Ribeiro et al.,
2004). This further demonstrates that only
a few subperineurial cells populate the
nerve and that they predominantly form
autocellular septate junctions as suggested
in the electron microscopic analyses.
Figure 2. Morphology of perineurial and subperineurial glial cells. To label individual cells or cell clones Flp expression was
induced in glial cells (repoFlp; repoGal4 UASactin::GFP; Gal80 FRT19A/FRT19A). Larval nervous systems were stained for Repo
expression (red) and GFP expression (green), and neurons were labeled with anti HRP (blue). A, MARCM cell clone of perineurial
glial cells in the third instar larval brain. Note the extensive cell protrusions generated by these cells (arrowhead). B, A large
MARCM clone consisting exclusively of perineurial cells in the abdominal part of the ventral nerve cord of a third instar larval brain.
Perineurial glial cells show many fine filopodia-like cell protrusions (arrowhead). C, Small flip-out clone of subperineurial glial
cells (repoGal4; UASflp; UAS⬎CD2 y⫹⬎mCD8GFP). In this confocal section, two of the nuclei can be identified. In contrast to the
perineurial glia, the subperineurial cells never form lateral filopodia-like extensions (arrowhead). D, D’, The large and flat subperineurial
glial cells are covered by an outer layer of glial cells, the perineurium. D’, In an orthogonal section, as indicated by the white line in D,
Repo-positive glial nuclei are seen apically to the GFP expressing glial cells (arrowhead). E, Projection of confocal stacks of a third instar
brain lobe stained for Repo (red), HRP (blue) and expression of NeurexinIV::GFP fusion protein that is confined to the septate junctions of
subperineurial cells. Scale bar, 85 ␮m. F, F’, CNS of a stage 16 embryo stained for Neurexin::GFP and Repo expression. Note that some
Repo-positive nuclei are located apically to the GFP expressing subperineurial cells.
Physiological role of the different
barrier layers
The different glial cells analyzed above
comprise the functional blood– brain barrier. The integrity of this
barrier can be measured after injection of labeled dextran molecules into the hemolymph (Bainton et al., 2005; Schwabe et al.,
2005). Furthermore, the use of differently sized dextran allows
addressing a possible size selectivity of the barrier. To determine
the kinetics of dextran uptake we used a Zeiss 5 Live LSM (see
Materials and Methods). This allowed us to directly follow and
quantify the dextran uptake in living embryos.
Stork et al. • Formation of Glial Barriers in Drosophila
J. Neurosci., January 16, 2008 • 28(3):587–597 • 591
glial layers seems to be intact in nrxIV mutants in addition to the lack of septate junctions, this suggests that most of the blood–
brain barrier function is conveyed by the
subperineurial glial cells and here in particular by the septate junctions formed between
these cells (supplemental Fig. 4, available at
www.jneurosci.org as supplemental material). Compared with neurexinIV mutants,
we also determined weaker uptake of 10 kDa
dextran into the CNS of moody mutant embryos which correlates with the reduced
number of septae found in the septate junctions of this mutant (Schwabe et al., 2005)
(Fig. 8M,P).
Septate junction independent blood–
brain barrier components
To test for other putative components of the
blood– brain barrier in addition to septate
junctions, we used differently sized labeled
dextran molecules. After injection of a labeled 70 kDa dextran into gcm mutants high
levels of fluorescence were immediately detected in the CNS (Fig. 8E). However, in contrast to the injection of a 10 kDa dextran, it
took ⬃20 min before comparable amounts
of fluorescence were detectable in mutant
Figure 3. Glial cells in the peripheral nervous system. To label individual cells or cell clones Flp expression was induced in glial neurexinIV embryos demonstrating that adcells of animals carrying a UAS⬎CD2 y⫹⬎mCD8GFP construct. Larval nervous systems were stained for HRP expression (blue), ditional barrier mechanisms exist (Fig. 8H,
Repo expression (red), and GFP expression (green). The top panel shows an overview of a ventral nerve cord with attached supplemental Fig. 5, available at www.jneuperipheral nerves. The boxed areas are shown in higher magnification in A–C. A, B, Peripheral nerve with a GFP labeled single rosci.org as supplemental material). Comwrapping glial cell. This glial cell spreads over at least 200 ␮m. The position of four orthogonal sections is indicated by small mercially available dextrans of 10 or 70 kDa
letters (a– d). The wrapping glial cells can form very thin processes that extend over long distances. C, C’, The upper nerve shows
are either neutral or negatively charged. To
a wrapping glia that has covered almost the entire fascicle. The lower nerve shows a perineurial glia clone. The arrowhead
compare the role of charge in penetration of
denotes a fine cell process. C’, Different confocal z-section of the same region as shown in C. Note the processes of the perineurial
glial cell that cover the nerve. The small letters indicate the position of the orthogonal sections (e, f ). Fine perineurial cell the blood– brain barrier we injected both
forms of 70 kDa in neurexinIV mutant emprocesses are indicated by an arrowhead (C’, f ). g, h, Two additional examples of perineurial glial cells.
bryos. As no difference was observed in these
experiments we conclude that charge is only a
When we injected a fluorescently labeled 10 kDa neutral dextran
minor factor regarding the tightness of the septate junction indepeninto stage 17 wild-type embryos, only little amount of fluorescence
dent component of the blood– brain barrier (data not shown).
could be detected in the ventral nerve cord with the laser settings
To test how the blood– brain barrier seals the nervous system
used in the experiment (Fig. 8A). Almost no increase in the level of
from even larger particles, we injected a 500 kDa anionic dextran
fluorescence can be detected within a 30 min observation period. To
with a size of ⬃26 nm in diameter (Luby-Phelps et al., 1986). In
genetically remove all components of the blood– brain barrier, we
comparison, GFP has a diameter of ⬃10 nm (Ormo et al., 1996).
used homozygous mutant glial cells missing ( gcm) embryos. In these
When gcm mutant embryos were injected, the neuropile immeanimals, all glial cells, except the midline glial cells, are routed toward
diately took up the injected dextran indicating that even large
a neuronal fate (Hosoya et al., 1995). When we injected labeled 10
particles can easily penetrate into the nervous system in the abkDa dextran into stage 17 homozygous mutant gcm embryos, high
sence of glial cells (Fig. 8 F). When we injected 500 kDa dextran
levels of fluorescence were detected within the nerve cord immediinto neurexinIV mutant animals, dye penetration was signifiately after injection and no increase in the level of fluorescence were
cantly reduced, corroborating the notion that not only septate
seen within 30 min (Fig. 8D,P). This reflects the complete loss of the
junctions of the subperineurial cells contribute to the physiology
blood– brain barrier in gcm mutants.
of the blood– brain barrier (Fig. 8 I). When we injected the 500
To address the function of the subperineurium we analyzed mukDa dye in moody mutant animals, dye penetration was further
tants known to affect the formation of septate junctions and comreduced (Fig. 8O). In summary, the formation of the blood–
pared their phenotypic consequences with those of gcm mutants.
brain barrier appears to depend mostly on the integrity of the
Mutants that remove important structural components of the sepseptate junctions formed by the subperineurial glial cell layer, but
tate junctions in epithelia such as neurexinIV mutants also show a
large molecules appear to be retained also by other barriers.
severe disruption of the integrity of the blood– brain barrier (Baumgartner et al., 1996; Schwabe et al., 2005; Strigini et al., 2006). Indeed,
Screening for genes required for blood– brain
fluorescently labeled 10 kDa dextran penetrated with very similar
barrier formation
kinetics into the ventral nerve cords of nrxIV or gcm mutant embryos
The above findings allow us to test for further components in(Fig. 8G,P). Because on the EM level the morphology of the outer
volved in the formation or maintenance of the blood– brain
592 • J. Neurosci., January 16, 2008 • 28(3):587–597
Stork et al. • Formation of Glial Barriers in Drosophila
barrier. In a first step we asked whether additional proteins associated with septate
junctions in epithelial cells are required to
set up a functional barrier. We injected labeled dextran molecules into embryos
lacking neurexinIV, neuroglian, nervana2,
contactin or coracle. For coracle and nervana2 mutants we obtained penetration
phenotypes comparable with what is seen
for mutant neurexinIV animals. Embryos
lacking neuroglian function have a slightly
less pronounced penetration phenotype
suggesting that septate junctions in the
nervous system are organized in a similar
manner as in the epithelial tissues (supplemental Fig. 6, available at www.jneurosci.org as supplemental material). Contactin was shown previously to be required for
normal barrier function and for the normal
cell surface expression of NeurexinIV
(Faivre-Sarrailh et al., 2004). In our hands, Figure 4. Sensory and motor axons project in distinct fascicles. Preparations of stage 16 nervous systems stained for sensory
and motor axons. Anterior is up. A, C, Embryo carrying the 43Gal4 insertion that directs GFP expression to many sensory neurons
we found that the blood– brain barrier of (green) and some neuropile glia in the CNS (ng) counterstained for the expression of the Fasciclin II protein (red) which is found
embryos homozygous for a contactin defi- on all motor axons as well as on some interneurons (in) in the CNS. Note that within the PNS sensory axons and motor axons are
ciency was less affected as in mutant neur- running in distinct trajectories (arrowheads). B, D, Embryo carrying a Fasciclin II GFP gene trap insertion (green) counterstained
exinIV embryos suggesting that Contactin with Mab 22C10 labeling the Futsch protein (red) that is expressed in the peripheral sensory neurons and their axons. A similar
does fulfill an auxiliary function in the separation of sensory axons and motor axons as in A and C can be seen. E, Third instar larvae expressing GFP in motor axons
blood– brain barrier as it was described for directed by a fasII:Gal4 driver (green). Motor axons are still found in one part of the nerve (sensory axons are labeled with 22C10;
the epithelium (Faivre-Sarrailh et al., 2004) Futsch protein, red; all axons are counterstained with anti HRP in blue). F, Third instar larval nerves expressing GFP in sensory
(supplemental Fig. 6, available at www.j- axons directed by the cha:Gal4 driver (green) do not intermingle with motor axons (red, anti-Fas II staining). The dotted line
indicates the position of the orthogonal section shown on the right.
neurosci.org as supplemental material).
In addition, we tested whether Claudinman and Brophy, 2005). Arthropods have not evolved saltatory
type proteins that, based on work in mammalian systems confer
conductance, but they are nevertheless in need for fast electrical
size selectivity to endothelial or epithelial barriers (Tepass, 2003;
conductance. In this respect it is not surprising that in marine
Furuse and Tsukita, 2006; Van Itallie and Anderson, 2006), are
shrimps myelin-like structures have been described previously
required for Drosophila blood– brain barrier formation. In the
(Davis et al., 1999; Weatherby et al., 2000). Drosophila follows
Drosophila genome six different Claudin-like proteins have been
two different and seemingly independent strategies to ensure fast
identified (Wu et al., 2004). Two of these proteins, Sinuous and
conductance. In some central neuronal networks large caliber
Megatrachea, have been shown to have a function in the epithelial
axons develop (Allen et al., 1998), whereas in the peripheral nerseptate junction dependent paracellular diffusion barrier (Behr et
vous system axons are insulated by several glial sheaths to ensure
al., 2003; Wu et al., 2004). Both proteins are also required for the
insulation. Initially, at the beginning of larval life, the different
integrity of the blood– brain barrier and homozygous mutant
sensory and motor axons are kept as separate fascicles within the
embryos allowed penetration of the 10 and 70 kDa dyes compasegmental nerves, suggesting there might be some degree of elecrable with moody mutant embryos, whereas penetration of the
trical cross talk within the different modalities. As the larva ma500 kDa dextran was not observed (for sinuous see Fig. 8 K, L,P).
tures, the inner wrapping glia starts to grow around single axons,
To test a possible contribution of the other four Claudin-like
which may allow more sophisticated movements of the wanderproteins we injected 10 kDa dextran into embryos carrying chroing larvae.
mosomal deficiencies removing these genes (for details, see MaWhereas the wrapping glia insulates individual axons do perterials and Methods). In all cases no disruption of the blood–
ineurial and subperineurial glia insulate the entire nervous sysbrain barrier was observed, suggesting that these Claudin-like
tem and set up the blood– brain-barrier. Genetic experiments
proteins are not needed for blood– brain barrier function (data
and ultrastructural studies have long indicated that septate juncnot shown).
tions provide the most effective part of this barrier (Auld et al.,
Discussion
1995; Baumgartner et al., 1996; Carlson et al., 2000). Indeed, the
subperineurial cell are formed early in development and these
A hallmark of any complex organized nervous system is its tight
cells are connected by septate junctions from late embryonic
insulation against high ion concentrations in the extracellular
stages onwards (Schwabe et al., 2005). Using Gal4 driver strains
fluids. This holds true for the mammalian blood– brain-barrier as
specific to the subperineurial cells as well as in vivo septate juncwell as for the much simpler barrier insulating the invertebrate
tion markers (Edenfeld et al., 2006), we confirm that during larval
nervous system where the high concentration of potassium in the
life the subperineurial cells do not divide but grow enormously
hemolymph would impede any regulated electrical conductance
large in size (Sepp et al., 2000; Silies et al., 2007). Septate junctions
(Carlson et al., 2000; Daneman and Barres, 2005).
formed by the subperineurial cells are mostly found in interdigFast neuronal conductance requires a tight electrical insulaitated zones of cell– cell contact. Cell division would likely require
tion of the axons and in the mammalian nervous system, myelin
and saltatory conductance evolved (Poliak and Peles, 2003; Sherdisintegration of septate junctions and thus result in a temporal
Stork et al. • Formation of Glial Barriers in Drosophila
J. Neurosci., January 16, 2008 • 28(3):587–597 • 593
The outermost glial cell layer is formed
by the perineurial cells. Although these
cells have long been described, their origin
is still a matter of debate (Edwards et al.,
1993; Schmid et al., 1999). In EM micrographs of late embryonic staged peripheral
nerves some perineurial cells can be detected apically to the subperineurial cells.
These glial cells divide during larval life and
generate a large number of fine cell protrusions that cover the subperineurial cells.
One function of the perineurium might be
to influence the development and/or the
tightness of the subperineurial layer. A
comparable cellular function has been attributed to the astrocytes in the mammalian nervous system (Abbott et al., 2006).
Alternatively, the perineurial glial cells
might provide a cellular basis for the response to injury (Smith et al., 1987). Unfortunately, we have to date no specific
driver strains that allow manipulation of
this glial cell population. Interestingly, a reverse relationship between subperineurial
and perineurial cells has been suggested
previously as subperineurial expression of
activated Ras or PI3K (phosphoinositide
3-kinase) resulted in an thickening of the
perineurial sheath (Lavery et al., 2007).
Additionally, the fray gene has been
shown to be required for normal axonal
ensheathment (Leiserson et al., 2000). Interestingly the mutant phenotype could be
rescued by expressing fray using three different Gal4 drivers (Leiserson et al., 2000).
After the analysis of the specificity of these
drivers (Mz317, subperineurial glia and
weak wrapping glia; Mz709, all glial cell
types; gliotactinGal4, subperineurial glia)
(data not shown), we conclude that fray is
expressed in subperineurial glia and controls axonal ensheathment of wrapping glia
in a noncell autonomous manner.
Given the different cellular barriers deFigure 5. Ultrastructural morphology of perineurial and subperineurial cells. A–G, The figure shows electron micrographs of scribed in this report, questions arise constage 16 embryonic nerves (A, B), first larval instar nerves (E–G), and third instar ventral nerve cord (C, D). A, In a peripheral nerve cerning the functional contributions of the
of a stage 16 nerve, perineurial (pg) and subperineurial glial cells (spg) are tightly associated with the axonal fascicles. The different layers. Here we have performed
wrapping glia (wg) does not ensheath individual axons. The boxed area is shown in magnification in B. B, No glial processes can kinetic studies that supported the imporbe detected within the fascicle. The axons are in direct contact with the subperineurial glia (spg) that forms a thin layer around the tance of the septate junctions in particular
fascicle (arrowheads). C, In the CNS of a third instar larvae the subperineurial glia (light blue shading) is characterized by its flat for small components. Animals lacking
appearance. Septate junctions can only be recognized in this glial layer (arrowhead). A thick neural lamella (nl) covers the nervous
septate junctions are as leaky to a 10 kDa
system. D, In the perineurial glial cell layer (light green shading), numerous cell protrusions can be seen (asterisk). These
dextran as animals lacking all glial cell layprocesses never invade into the subperineurial layer. E, In a first instar larval nerve, septate junctions are formed by the subperineurial cells. The image corresponds to the dotted area in F. F, Only few perineurial glial cells are found along the nerve. They do ers. However, when it comes to larger molnot fully cover the subperineurial cells as they do in later developmental stages. The wrapping glia has not yet started to ecules, the relevance of the other cell layers
individually ensheath every axon. G, The different glial cells are highlighted by shading. nl, Neural lamella; pg, perineurial glia; becomes obvious. Although a 500 kDa dextran can easily penetrate into the nervous
spg, subperineurial glia; sj, septate junction; wg, wrapping glia; ax, axons. Scale bars: 1 ␮m.
system of a glial cells missing embryo, its
leakage into the nervous system of a neuropening of the blood– brain barrier, which would be deleterious
exinIV mutant lacking septate junctions is greatly reduced. Thus,
for the animal. This is in agreement with previous findings that
the other layers contribute to the function of the blood– brain
Gliotactin expressing cells, forming septate junctions, do not dibarrier. Because a continuous perineurium is not fully formed in
vide during larval live (Sepp et al., 2000; Schulte et al., 2003,
first instar larvae, the barrier function has to be assigned to the
2006).
neural lamella and the inner glial layer. There are several reports
594 • J. Neurosci., January 16, 2008 • 28(3):587–597
Stork et al. • Formation of Glial Barriers in Drosophila
showing that the neural lamella can act as
an efficient filter for heavy metal ions
(Carlson et al., 2000). Possibly, large molecules such as the 500 kDa dextran are also
trapped in this ECM. Alternatively, large
particles are stopped by the diffusion barrier established by the normal cell– cell
contacts between subperineurial cells and
inner glial cell types like wrapping glia in
the peripheral nerves and cortex and neuropile glia in the CNS.
The diffusion barrier provided by glial
cells or epithelial sheaths is generated by
special junctional complexes that help to
tightly associate the involved cells. Drosophila epithelia as well as glial cells are
characterized by septate junctions (Tepass
and Hartenstein, 1994). Quite similar
structures are also found at the mammalian
paranodal junctions, which provide the
structural basis for the tight electrical insulation of the nerve. A core component of
the mammalian axoglial septate junctions
is the NeurexinIV homolog Caspr that together with its binding partners, Contactin
and Neurofascin155, sets up a tripartite adhesion complex at the paranode (Bhat et
al., 2001; Girault and Peles, 2002; Poliak
and Peles, 2003; Sherman and Brophy,
2005).
The function of this complex appears
conserved in Drosophila, although there are
some notable differences. The Caspr homolog NeurexinIV is expressed by glial
cells as are Contactin and the Neurofascin155 homolog Neuroglian (FaivreSarrailh et al., 2004; Banerjee et al., 2006).
As a consequence, in the fly septate junctions are formed between glial cells,
whereas they are formed between neuronal
and glial membranes in the mammalian
system. The Caspr/Contactin/Neurofascin155 complex seals the paranodal junc- Figure 6. Three glial cell layers are present in the larval nerve. A, Electron micrograph of a third larval instar peripheral nerve.
tion and a similar function has been attrib- The neural lamella (nl) has the same size throughout the nerve circumference. Below are the perineurial glial cells (pg, one cell is
uted to this protein complex in the highlighted by light blue shading) that show various indentations and profiles of small cell protrusions. The fascicle is encircled by
invertebrate blood– brain barrier (Bhat, one subperineurial glial cell that forms only short processes toward the axon fascicle (spg, arrows, the cell is labeled in blue). The
2003). Here, we found a less pronounced subperineurial glia forms autocellular septate junctions (boxed area and magnification; white arrowhead points to septate
junctions; black arrowhead points to septate junction-free cell– cell contact). Within the fascicle, usually two to three glial cell
function of Contactin compared with profiles can be detected (one profile is highlighted in red). B, Third instar larval nerves were stained for HRP (blue), Repo
NeurexinIV for the blood– brain barrier es- expression (red), and Dlg:GFP expression (green). Scale bar, 1 ␮m. B, C, Lateral and orthogonal (C) view of a larval nerve
tablishment, corroborating findings made expressing a Dlg:GFP fusion protein under the control of the SPG:Gal4 driver. GFP expression accumulates at the septate junctions
in embryonic epithelia (Faivre-Sarrailh et formed by the subperineurial glial cell.
al., 2004).
Another prominent component of the
component of tight junctions of brain endothelial cells. claudin5
junctional complexes are the Claudin proteins. In mammals,
mutant mice show no structural or ultrastructural deficits, but
members of these four transmembrane domain proteins are ashave an impaired blood– brain barrier (Morita et al., 1999; Nitta
sociated with tight junctions that are often considered to be funcet al., 2003). The association of Claudins integrated in opposing
tionally equivalent to the invertebrate septate junctions (Furuse
membranes is thought to provide pores that can control the paraand Tsukita, 2006). In Drosophila two Claudin-like proteins have
cellular diffusion of small molecules. Although Drosophila Sinubeen described to be required for formation of normal epithelial
ous and Megatrachea clearly contribute to the barrier function, it
barrier formation (Behr et al., 2003; Wu et al., 2004). Here, we
is inconceivable that fly Claudins traverse the 20 nm wide septate
show that both Sinuous and Megatrachea are also needed for the
gap to form a Claudin pore as it is discussed for the vertebrate
establishment of normal blood– brain barrier formation. SimiClaudins (Furuse and Tsukita, 2006; Van Itallie and Anderson,
larly, it was shown previously that mammalian claudin5 is a major
Stork et al. • Formation of Glial Barriers in Drosophila
J. Neurosci., January 16, 2008 • 28(3):587–597 • 595
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